Dynamics of human neutrophil aggregation evaluated by flow cytometry

Area of Science:

• Hematology
• Immunology
• Biophysics

Background:

• Neutrophil aggregation is crucial in inflammatory responses.
• Accurate monitoring of neutrophil aggregation is essential for understanding immune cell dynamics.
• Existing methods for measuring neutrophil aggregation can be complex or indirect.

Purpose of the Study:

• To develop and validate a simple flow cytometry-based particle counting technique for direct monitoring of neutrophil aggregation.
• To introduce and define key aggregation parameters based on particle count changes.
• To investigate the influence of initial cell concentration and shear forces on neutrophil aggregation kinetics.

Main Methods:

• Development of a particle counting technique using flow cytometry to measure changes in total particle count (%PA(T)) and singlet neutrophils (%PA(S)).
• Comparison of flow cytometry results with traditional microscopy methods.
• Stimulation of neutrophils with N-formyl-methionyl-leucyl-phenylalanine (FMLP) to induce aggregation.
• Analysis of aggregation parameters including latent time (tl), forward rate (vf), and aggregation duration under varying initial cell concentrations (N(o)) and stir speeds.

Main Results:

• Flow cytometry particle counting method yields results comparable to microscopy.
• Aggregation parameters can be accurately assessed solely by monitoring total particle count changes.
• Neutrophil aggregation induced by FMLP exhibits a short latent time (4 ± 1.5 sec) independent of N(o) or stir speed.
• The forward rate of aggregation (vf) is directly proportional to N(o) and stir speed.
• Aggregation plateaus for at least 60 seconds and shows partial reversal within 7.5 minutes.
• Aggregation extent and duration are inversely related to stir speed above a critical threshold.

Conclusions:

• The developed flow cytometry particle counting technique provides a simple and effective method for quantifying neutrophil aggregation.
• Aggregation parameters are significantly influenced by initial cell concentration and shear forces, suggesting subpopulations of neutrophils with varying aggregation efficiencies.
• This method facilitates direct monitoring and comparison of neutrophil aggregation dynamics in various physiological and pathological conditions.